Microfluidic probe with bypass and control channels

ABSTRACT

A microfluidic probe includes a probe head with a processing surface that includes a first aperture and a second aperture. The probe further includes a liquid injection channel, which leads to the first aperture, and a liquid aspiration channel, which extends from the second aperture. The probe also includes a bypass channel, arranged so as to fluidly connect the liquid injection channel to the liquid aspiration channel, as well as a control channel. The latter fluidly connects to the bypass channel, hence forming a junction therewith, so as to define two portions of the bypass channel. These portions includes: a first portion that extends from the junction to the liquid injection channel; and a second portion that extends from that same junction to the liquid aspiration channel. The invention is further directed to methods of operation of a probe as described above, to process a surface.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/588,612 filed 6 May 2017, the complete disclosure of which isexpressly incorporated herein by reference in its entirety for allpurposes.

BACKGROUND

The invention relates in general to the field of microfluidics,microfluidic probe systems and microfluidic probe heads equipping suchsystems. In particular, it is directed to a microfluidic probecomprising liquid injection and aspiration channels, connected by abypass channel, which is itself connected by a control channel.

Microfluidics deals with the precise control and manipulation of smallvolumes of fluids that are typically constrained to micrometer-lengthscale channels and to volumes typically in the sub-milliliter range.Prominent features of microfluidics originate from the peculiar behaviorthat liquids exhibit at the micrometer length scale. Flow of liquids inmicrofluidics is typically laminar. Volumes well below one nanoliter canbe reached by fabricating structures with lateral dimensions in themicrometer range. Microfluidic devices generally refer tomicrofabricated devices, which are used for pumping, sampling, mixing,analyzing and dosing liquids. A microfluidic probe is a device fordepositing, retrieving, transporting, delivering, and/or removingliquids, in particular liquids containing chemical and/or biochemicalsubstances. For example, microfluidic probes can be used in the fieldsof diagnostic medicine, pathology, pharmacology and various branches ofanalytical chemistry. Microfluidic probes can also be used forperforming molecular biology procedures for enzymatic analysis,deoxyribonucleic acid (DNA) analysis and proteomics.

A number of failure scenarios may occur when processing a surface withsuch microfluidic probes.

SUMMARY

According to a first aspect, the present invention is embodied as amicrofluidic probe. The device comprises a probe head with a processingsurface that comprises a first aperture (i.e., an injection aperture)and a second aperture (i.e., an aspiration aperture). The probe furtherincludes a liquid injection channel, which leads to the first aperture,and a liquid aspiration channel, which extends from the second aperture.Remarkably, the probe also comprises a bypass channel, arranged so as tofluidly connect the liquid injection channel to the liquid aspirationchannel, as well as a control channel. The latter fluidly connects tothe bypass channel, hence forming a junction therewith, so as to definetwo portions of the bypass channel. These portions includes: a firstportion that extends from said junction to the liquid injection channel;and a second portion that extends from that same junction to the liquidaspiration channel. The probe is preferably designed so as to allow ahydrodynamic flow confinement of processing liquid injected through thefirst aperture and aspirated from the second aperture.

The above structure makes the MFP technology robust against partial orcomplete blockage of one or several of its apertures, e.g., when the MFPhead is in contact with the surface or while scanning the surface withthe head. That is, in case of failure, the injected processing liquidcan be diverted through the bypass channel, instead of leaving theprobe, assuming a suitable liquid/pressure flow is applied to thecontrol channel. There are indeed circumstances where one wants to avoidthe processing liquid to uncontrollably escape the probe as thistypically leads to a loss of confinement of the processing liquid andmay then contaminate the immersion liquid and subsequently thesubstrate. As a further advantage, the above probe can be operated inconstant flow mode or constant pressure mode.

In embodiments, the hydraulic resistance of the first portion of thebypass channel is larger than the hydraulic resistance of the secondportion of the bypass channel, which makes it possible to limit the flowthat can pass from the control channel through the injection aperture(e.g., in case the operating distance becomes excessively large). Forexample, the hydraulic resistance of the first portion may be between 2and 100 times larger than the hydraulic resistance of the secondportion.

In preferred embodiments, the first portion of the bypass channel has anaverage cross-section that is smaller than an average cross-section ofthe second portion of the bypass channel. This way, the resistances ofthe two channel portions can be easily varied, without requiring changein the surface material properties.

Yet, the first portion and the second portion of the bypass channelshall preferably have a same depth (which simplifies the fabricationprocess), while the first portion will have, on average, a smaller widththan the second portion.

Preferably, the first portion of the bypass channel has a length that islarger than the length of the second portion of the bypass channel, soas to achieve a larger hydraulic resistance for the first portion.

Whereas the bypass channel portions may have different resistances, theliquid injection channel, the liquid aspiration channel and the controlchannel preferably have, each, a constant hydraulic resistance alongtheir main channel extensions.

However, in preferred embodiments, the hydraulic resistance of thecontrol channel is made smaller than the hydraulic resistance of each ofthe injection channel and the aspiration channel, to allow sufficientaspiration flow rates in practice.

Preferably, the bypass and control channels are provided directly in theprobe head, to allow faster reaction times in case of failures. That is,each of the liquid injection channel, the liquid aspiration channel, thebypass channel and the control channel extends within a body of theprobe head, so as for the bypass channel to fluidly connect, within thebody, the liquid injection channel to the liquid aspiration channel. Invariants, the bypass and control channels are provided in a bypassmodule, outside the probe head. Such an “off-chip” (or “off-head”)configuration makes it possible to re-use existing probe heads.

In each case, the present probes may notably be equipped with probeheads of the so-called “vertical” type or, in variants, of the“horizontal” type. And in each case, the fabrication of the heads can bekept simple, involving a few layers of materials.

For example, in embodiments relying on a horizontal probe head thatincludes the bypass and control channels, the head may comprise twolayers, i.e., a control layer and a routing layer, where a bottom faceof the control layer covers a top face of the routing layer. Theprocessing surface is defined by a bottom face of the routing layer,opposite to the top face thereof, whereby the first and second aperturesare, each, defined on the bottom face of the routing layer. Moreover,the routing layer comprises a first pair of through-vias extendingthrough a thickness thereof, so as to form segments of the liquidinjection channel and the liquid aspiration channel, in fluidcommunication with the first aperture and the second aperture,respectively. The routing layer further comprises the bypass channel,which is defined on the top face of the routing layer. The control layercomprises a through-via extending through a thickness thereof, so as toform a segment of the control channel. The control layer furtherincludes a second pair of through-vias extending through a thicknessthereof, so as to form additional segments of the liquid injectionchannel and the liquid aspiration channel, respectively, in fluidcommunication with said first pair of through-vias, respectively.

In embodiments where the probe head is configured as a vertical probehead, the latter preferably comprises two layers (at least) ofmaterials. Each of the first segment of the liquid injection channel andthe first segment of the liquid aspiration channel are grooved on one ofthese two material layers and closed by the other one of the other twomaterial layers. The bypass and control channels may further be groovedon the same layer as the first segments of the injection and aspirationchannels.

In embodiments where the bypass-concept is implemented outside the probehead, a first segment of the liquid injection channel and a firstsegment of the liquid aspiration channel may be defined on (or in) theprobe head, so as to be in fluid communication with the first apertureand the second aperture, respectively. However, the probe furthercomprises a bypass module, which is distinct from the probe head,wherein the bypass module comprises the bypass channel and the controlchannel, as well as a second segment of the liquid injection channel anda second segment of the liquid aspiration channel. The bypass channelfluidly connects, within the bypass module, the second segment of theliquid injection channel to the second segment of the liquid aspirationchannel. For completeness, the second segment of the liquid injectionchannel and the second segment of the liquid aspiration channel need bein fluid communication with the first segment of the liquid injectionchannel and the first segment of the liquid aspiration channel,respectively.

Preferably, the probe head is fixed to the bypass module, and the probehead comprises through-vias, so as for the second segment of theinjection channel and the second segment of the aspiration channel to bein fluid communication with the first segment of the injection channeland the first segment of the aspiration channel, respectively.

In embodiments, the processing surface comprises a set of two or moresecond apertures, including said second aperture, wherein each of thetwo or more second apertures is arranged at a distance from the firstaperture on the processing surface. In such cases, the probe comprises:

-   -   a set of two or more liquid aspiration channels, including said        liquid aspiration channel, wherein each of the two or more        liquid aspiration channels extends from a respective one of the        second apertures;    -   a set of two or more bypass channels, including said bypass        channel, each arranged so as to fluidly connect the liquid        injection channel to a respective one of the liquid aspiration        channels; and    -   a set of two or more control channels, including said control        channel, each fluidly connecting to a respective one of the two        or more bypass channels, so as to allow processing liquid        injected via the injection channel to be diverted through the        bypass channels, if needed.

In embodiments, the second aperture comprises a slit, shaped so as topartly extend around the first aperture on the processing surface. Yet,the first aperture is not completely surrounded by the slit on theprocessing surface.

In embodiments, the probe comprises a plurality of bypass channels,including said bypass channel, each arranged so as to fluidly connectthe liquid injection channel to the liquid aspiration channel. Havingmultiple bypass channels allows a gradual diversion of the processingliquid, when necessary. It further allows the device to have differentworking points, i.e., different bypass thresholds can be set, whichmakes it possible to cope with different failure scenarios with a samedevice, operated in a fully passive mode.

Preferably, the probe is configured to operate in one or each of twomodes, the latter including:

-   -   a constant liquid flow mode, wherein a constant liquid flow is        applied to each of the liquid injection channel, the liquid        aspiration channel, and the control channel; and    -   a constant pressure actuation mode, wherein a constant pressure        is applied to each of the liquid injection channel, the liquid        aspiration channel and the control channel.

According to another aspect, the invention can be embodied as a methodof operating a probe such as described above. Basically, this methodcomprises: positioning the probe head in proximity with a sample surfaceto be processed, so as for the processing surface to face the samplesurface; and injecting processing liquid via the first aperture whileaspirating liquid from the second aperture, to process the samplesurface.

In typical applications, the probe head is positioned in proximity withan immerged sample surface. I.e., an immersion liquid covers thatsurface, so as for the probe head to be at least partly immersed in theimmersion liquid. As a result, some of this immersion liquid getstypically aspirated from the second aperture. Preferably, the liquidinjection and aspiration are performed so as to maintain a hydrodynamicflow confinement of injected liquid between the injection aperture andthe aspiration aperture.

Processing the surface may lead to block one or each of the firstaperture and the second aperture, due to a proximity of the probe headwith the sample surface processed. As per the design of present probes,the processing liquid injected via the injection channel maynevertheless pass through the bypass channel and be aspirated via theaspiration channel.

In preferred embodiments, the present probes are used as passivesystems. However, in variants, they may be dynamically controlled, whichmay basically require to adjust a liquid flow rate or a liquid pressurein the control channel, in operation. Still, one understands thatadjusting the liquid flow rate in the control channel likely impacts theliquid pressure(s) in other channels and, conversely, adjusting thepressure in the control channel typically impacts the various liquidflow rates.

In passive systems, the liquid flow rate or the liquid pressure may beadjusted (e.g., once for all) prior to positioning the probe head inproximity with the sample surface. Then, the liquid flow rate or theliquid pressure is kept constant in the control channel, while injectingthe processing liquid via the first aperture and aspirating liquid fromthe second aperture to process the sample surface.

Devices, apparatuses, systems and methods embodying the presentinvention will now be described, by way of non-limiting examples, and inreference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a microfluidic probe, according to embodiments. The probecomprises bypass and control channels provided directly in the probehead;

FIG. 2A shows a side view of a probe head as involved in FIG. 1—acorresponding circuit of hydraulic resistances of the various liquidflow paths is symbolically superimposed on the view.

FIG. 2B depicts the same and additionally shows constraints between thehydraulic resistances, as involved in embodiments;

FIGS. 3-6 show side views of the same probe head, in operation, andillustrate how the bypass concepts operates in case of failurescenarios, as involved in embodiments;

FIGS. 7-8 illustrate variants to the probe head of FIG. 1, which involvemultiple bypass and/or control channels;

FIGS. 9-10 illustrate variants to the microfluidic probe system of FIG.1, wherein the bypass channel is provided in a module distinct from theprobe head, here assumed to be of the “vertical” type;

FIG. 11A shows a photograph of a layer of vertical probe head, whichintegrates the bypass and control channels, according to embodiments;

FIG. 11B shows a magnified portion of the channel structure about thebypass for the head of FIG. 11A;

FIG. 11C depicts an exploded, 3D view of the vertical head of FIG. 11A,showing how channels can be grooved on a lower layer and closed by anupper layer, as in embodiments;

FIG. 12 is a 3D (partial) view of a microfluidic device that includes a“horizontal” probe head, according to embodiments;

FIG. 13 illustrates the assembly of three layers (depicted incross-section) to obtain a multilayer, horizontal probe head, which canbe used in a device as depicted in FIG. 12;

FIGS. 14A-C schematically depict views of different types of aperturesthat can be formed on processing surfaces of probe heads, as involved inembodiments;

FIG. 15 is a photograph (taken with an inverted microscope through aglass slide) of the processing surface of a horizontal probe head,having apertures designed as in FIG. 14C, as involved in embodiments.The photograph shows a (whitish) liquid flow that is hydrodynamicallyconfined between the injection aperture and the partly surroundingaspiration aperture; and

FIG. 16 is another photograph, illustrating how the NFP head of FIG. 15can be scanned across a surface to deposit cells, according toembodiments.

The accompanying drawings show simplified representations of devices orparts thereof, as involved in embodiments. Technical features depictedin the drawings are not necessarily to scale. Similar or functionallysimilar elements in the figures have been allocated the same numeralreferences, unless otherwise indicated.

DETAILED DESCRIPTION

A unique feature of microfluidic probes (scanning, non-contacttechnology) is the possibility to localize the processing liquid on animmersed substrate, due to a hydrodynamic flow confinement (HFC) of theprocessing liquid in the immersion liquid. Ideally, what is needed forsuch a scanning probe technology to reliably operate is: (i) a constantprobe-to-surface distance during the scanning, which, ideally, requiresa surface free of substantial topographical variations; and (ii) noparticulate contamination of the liquids both in the processing andimmersion liquid, to avoid clogging the channels.

In practical implementations though, the probe-to-substrate distance (or“operating distance”) can vary when scanning the probe head over thesubstrate. Typical variation amplitudes are of 0.1 mm. Such variationsmay result in temporary blocking one or several of the apertures of thedevice. This, as the present Inventors observed, may cause a break-downof the localization of the liquid flow, resulting in a contamination ofthe substrate by the processing liquid. In addition, particulates in theprocessing/immersion liquid flowing in the injection/aspiration channelscan perturb the flow conditions, in particular, during extended periodsof operation.

The present Inventors have therefore designed concepts of microfluidicprobes (or MFPs) and operation methods that improve the robustness ofthe MFP technology. In particular, such concepts make the MFP technologymore robust against partial or complete blockage of one or severalapertures of the devices.

In reference to FIGS. 1-11, an aspect of the invention is firstdescribed, which concerns a microfluidic probe 1, 1 a-1 f. Microfluidicprobes are sometimes referred to as microfluidic (or MFP) devices,apparatuses or systems in the literature. A microfluidic probes notablycomprises a probe head (or MFP head), designed to come in contact withand process a sample surface. In addition, it typically comprisesadditional components needed to operate the MFP head, such as tubes,tubing ports, liquid tanks, pressure or vacuum sources, valves,additional MFP modules, etc.

As depicted in the accompanying drawings, the present probe conceptincludes a probe head 10, 10 a-10 h, which exhibits a processing surface11, onto which are defined a first aperture 112 and a second aperture114. The processing surface 11 typically forms a boundary of the probehead, e.g., a face of the head, meant to face the surface of the sampleto be processed, in operation.

As usual in the art, the probe comprises a liquid injection channel 12,which leads to the first aperture 112, as well as a liquid aspirationchannel 14, which extends from the second aperture 114. Thus, theapertures 112, 114 can be respectively regarded as a liquid injectionaperture and a liquid aspiration aperture. The injection channel is usedto inject liquid toward the surface S to be processed, i.e., to ejectliquid from the first aperture 112, whereas the aspiration channel isused to (re-)aspirate liquid from the surface 5, in operation. Thisassumes that the probe is otherwise configured to allow liquid injectionand liquid aspiration via the channels 12, 14.

Remarkably here, the probe further includes a bypass channel 15, whichis arranged so as to fluidly connect the liquid injection channel 12 tothe liquid aspiration channel 14. I.e., the bypass channel 15 physicallyconnects, directly, to each of the injection channel 12 and theaspiration channel 14, so as to form respective junctions J1, J2therewith, as depicted in FIG. 1.

In addition, the probe comprises a control channel 16, which fluidlyconnects to the bypass channel 15, hence forming a junction J3therewith. I.e., there are at least three junctions J1, J2, J3 in total,one J3 formed between the control channel 16 and the bypass channel 15,in addition to the two junctions J1, J2 formed at the ends of the bypasschannel 15 with the channels 12, 14. The junction J3 is typicallylocated between the injection channel 12 and the aspiration channel 14,e.g., between the two junctions J1, J2.

The junction J3 formed between the control channel 16 and the bypasschannel 15 implies distinct channel portions 151, 152 for the bypasschannel 15, formed on each side of this junction J3. A first channelportion 151 extends from the junction J3 to the injection channel 12,while a second portion 152 extends from that same junction J3 to theaspiration channel 14. Thus, the two portions 151, 152 potentiallyenables fluid communication between each of the channels 12, 14 and arespective portion 151, 152 of the bypass channel 15 it connects to.

The extent to which fluid communication is enabled between the channels12, 14 and their respective portions 151, 152 is governed by a number ofparameters, as discussed below in detail. Yet, this can be controlled(at least partly) thanks to the control channel 16, assuming that theprobe is configured to apply a liquid flow (or pressure) to this channel16.

If necessary, more than one control channel may be provided, eachconnecting to a same or a respective bypass channel, in order to adaptthe bypass properties or ensure sufficient control on a bypass channel,as latter discussed in reference to FIGS. 7-8. However, a single bypasschannel and a single control channel already makes it possible to copewith various failure scenarios in practice.

The present probes are preferably configured so as to allow hydrodynamicflow confinement of the injected liquid, as assumed in most of theembodiments described below.

Thanks to the bypass channel 15 between the injection and aspirationchannels 12, 14 and the control channel 16 connected thereto, thepresent concept makes the MFP technology robust against partial orcomplete blockage of one or several of the apertures of the head, e.g.,when bringing the MFP head in contact with the surface processed orwhile scanning this surface with the head.

In normal operation (as assumed in FIG. 1), no processing liquid (asinjected through channel 12) should pass through the entire bypasschannel 15. To enable this normal mode of operation, the control channel16 need to connect the bypass channel 15 at a junction J3 that isdistinct from the two outer junctions J1, J2. That is, two well-definedportions 151, 152 of the bypass channel 15 need be defined on each sideof the junction J3. In other words, each of the hydraulic resistances R4and R5 (associated to respective portions 151, 152, see FIGS. 2A, 2B) isstrictly greater than zero. This way, the pressure at the junction J3can be matched to the pressure at the junction J1 between the injectionchannel 12 and the bypass channel 15, by varying the pressure in thecontrol channel 16. This results in a stagnation of the liquid flow,i.e., no additional liquid may enter the bypass channel 15 from theinjection channel 12 across the portion 151.

Yet, in case of failure (e.g., blockage of one or each of theapertures), the processing liquid can be passed through the entirebypass channel 15 (i.e., though both portions 151 and 152), instead ofleaving the probe, as illustrated in FIGS. 3-5. There are indeedcircumstances where one wants to avoid the processing liquid to escapethe probe as this may typically lead to a loss of confinement of theprocessing liquid, which may then contaminate the immersion liquid andthe substrate, as noted earlier.

The transition threshold between the normal operation and failure modecan be set by suitably adjusting the flow rate/pressure in the controlchannel 16 or the value of the hydraulic resistance R4+R5 of the bypasschannel 15.

As it may further be realized, the present MFP concepts are compatiblewith a constant flow mode or a constant pressure mode of operation,which allow, each a fully passive operation of the probe head. That is,the probe can be operated in constant flow mode or in constant pressuremode. In constant pressure mode, liquid tanks would typically need beconnected to the injection channel 12, the control channel 16 and theaspiration channel 14 (not shown). The pressure and vacuum levelsapplied 41-43 to said liquid tanks remain constant. In constant flowmode, a constant flow rate is maintained in the injection channel 12,the control channel 16 and the aspiration channel 14, by, e.g.,employing a dedicated syringe pump to effect flow in these channels. Apassive compensation for failures as described above occurs in bothmodes of operations in essentially the same manner.

Yet, the present approach allows active control of one or more of thevarious liquid flows involved. Thus, fully passive or fully activecontrol schemes can be contemplated. Now, various intermediate schemescan be contemplated, involving only a partial control of the liquidflows (e.g., in the control channel 16 only). In addition, the presentMFP concepts are further compatible with various head configurations andaperture designs, as exemplified in FIGS. 1, 7-14. Moreover, the bypassand control channels can be implemented directly on the head or in anexternal module 30 (“off-head”). All this is discussed below in detailin reference to specific embodiments.

Referring now more particularly to FIGS. 2A and 2B, we note that thereare at least two flow paths between the junctions J1, J2 formed betweenthe bypass channel 15 and the injection channel 12 and the aspirationchannel 14. Additional flow paths may exist in case more bypass channelsare implemented or if the bypass channel 15 branches into severalchannels which are connected to the aspiration channel 14 at differentlocations. This would allow to have multiple bypass channels, which arededicate to alter the flow path in case of specific failure events. Inthe most basic design featuring a single bypass channel, one flow pathoccurs along the bypass channel 15, which has a hydraulic resistance ofR4+R5. A second flow path occurs between the apertures 112, 114, acrossthe space between the probe head 1 and the surface S of the sampleprocessed; it has a hydraulic resistance of R6+R8+R7. When typicaldesign parameters are used for the channels 12, 14, 16 and apertures112, 114, the hydraulic resistances of those two flow paths are in thesame range, so that the entire flow of injected liquid can be passedthrough either of the two flow paths without significant deviations ofthe operating pressures. The resistances R8, R9, and R10 are resistancesbetween the probe head 1 and the surface S. The resistance R8, forexample, varies with the distance of the probe 1 from the surface S.Yet, the hydraulic resistance of the bypass channel 15 may be adjustedto a desired, normal operating distance. Conversely, the operatingdistance could also be adjusted, in some extent, depending on theresistance of the bypass channel 15.

At a standard operating distance (between the probe and the surface S),no liquid flowing through the injection channel 12 should enter thebypass channel 15 in order to minimize consumption of reagents.Consequently, typically an inexpensive buffer is injected in the controlchannel 16 to cause stagnation of flow in the portion 151 (between J1and J3).

Now, when the distance between the probe 1 and the surface S of thesample deviates from the standard operating distance (as illustrated inFIGS. 3-6), the bypass and control channels cause the composition of theflow through the two flow paths evoked above to passively reconfigure.This, in turns, compensate for undesired consequences of excessivelysmall and excessively large operating distances.

In case the operating distance becomes smaller than the standardoperating distance (FIGS. 3-5), the value of R6+R7+R8 becomes largerthan that of R4+R5 and the liquid flowing in the injection channel 12 ispassively diverted to follow the path of lower resistance through thebypass channel 15. The proportion of the flow through the injectionchannel 15 that is redirected through the bypass channel 16 varies,depending on the operating distance. In case of full contact between theprobe and the surface S (FIG. 5) or in case of blockage of the injectionaperture 112 (FIG. 4) or the aspiration aperture 114 (FIG. 3), theentire processing liquid flow (through injection channel 12) isredirected through the bypass channel 15 to enter the aspiration channel14. Thus, leakage of the processing liquid (e.g., into immersion liquid)can be prevented.

In case the operating distance becomes greater than the standardoperating distance (as assumed in FIG. 6), the value of R6+R7+R8 becomessmaller than that of R4+R5 and the liquid flowing in the control channel16 gets passively diverted to flow through the injection aperture 112.Hence, the flow through the injection aperture 112 increases and thevolume occupied by the injected liquid in the space between the probe 1and the surface S of the sample protrudes further downwards andpartially compensates for the large operating distance.

The resistances in the injection channel 12, control channel 16 andaspiration channel 14 allow a precise control of the respective flowrates, e.g., by controlling the pressure in liquid tanks connected tothose channels. The resistance values R1, R2 and R3 therefore depend onthe desired range of flow rates and precision. Still, since the flowthrough the injection channel 12 and the control channel 16 willtypically be comparable (if not equal) during standard operation, R3 canadvantageously be made smaller (e.g., about five times smaller) thaneach of R1 and R2, to enable sufficient aspiration flow rates. We note,however, that the resistance R3 need not systematically be smaller thanR1 or R2, e.g., when employing a syringe pump to effect flow in thesechannels 12, 16.

As further shown in FIG. 2B, the hydraulic resistance R4 of the bypasschannel portion 151 is preferably made larger than the hydraulicresistance R5 of the second portion 152. As one may realize, this makesit possible to limit the flow that can pass from the control channel 16through the injection aperture 112 in case the operating distancebecomes larger than the standard operating distance. In such a scenario,if R4 is too small, the flow of liquid injected through the injectionaperture 112 can be so high that the injected liquid is not aspiratedback through the aspiration aperture 114, hence contaminating thesurrounding immersion liquid.

Whenever possible, the head should further be designed so as to preventleakage of the injected liquid to the surrounding immersion liquid.Therefore, R4 is preferably designed to be larger than R5, in which casethe hydraulic resistance of the bypass channel 15 is mainly impacted byR4. This prevents leakage in case of excessively large and excessivelysmall operating distances. Still, the hydraulic resistance R5 of thesecond channel portion 152 is required to be able to create stagnationof flow (no flow condition across the first portion of the bypasschannel 151).

To that aim, the hydraulic resistance R4 of the channel portion 151 maytypically be between 2 and 100 times larger than the resistance R5 ofthe second channel portion 152. Yet, a ratio that is between 3:1 and20:1 (e.g., 10:1) for R4:R5 was experimentally shown to be most suitablein practice. In variants, however, one may in fact specifically want tohave R4<R5, e.g., in order to allow a liquid flow from the controlchannel 16 to enter the injection channel 12, so as to expand the volumeof the hydrodynamic flow confinement, e.g., in case of large operatingdistances.

It is worth to remind that the hydraulic resistance of a channel (or achannel portion) is essentially determined by intrinsic feature of thischannel (e.g., like dimensions, surface material, etc.). However, thehydraulic resistance typically scales with the flow rate, the pressure,the viscosity of the liquid, etc. Nevertheless, it remains that thebypass channels (or channel portions) may be suitably designed (e.g.,dimensioned) so as to maintain certain desired relations between theresistances (e.g., to make sure that that R4>R5 and/or R3<R1, R2), forusual liquids and standard liquid flow rate/pressure, as used in MFPs inpractice.

As illustrated in FIGS. 11A-11B, the desired relations between thevarious hydraulic resistances may notably be obtained by adapting theaverage cross-sections of the channels. For instance, in the embodimentof FIGS. 11A-11C, the probe head 10 e (here of a vertical type) isdesigned such that the first portion 151 of the bypass channel 15 has anaverage cross-section that is smaller than the average cross-section ofthe second portion 152 of the bypass channel 15. Varying the averagecross-section of the channel portions allows to vary their hydraulicresistances, without requiring any change of surface materialproperties.

For example, and as illustrated in FIGS. 11A-11C, one may vary thewidths of the channel portions, yet without varying the channel depth,which makes it relatively simple from the fabrication view point. I.e.,in FIGS. 11A-11C, the first portion 151 and the second portion 152 ofthe bypass channel 15 have a same depth, while the first portion 151has, on average, a smaller width than the second portion 152. Only thewidth of the channel portions 151, 152 is varied, whereas the etch depthcan be kept constant.

As evoked earlier, the present MFP probes 1, 1 a-1 f shall preferably beconfigured to allow a hydrodynamic flow confinement (HFC) of theprocessing liquid injected through the aperture 112 and aspirated fromaperture 114. Generally speaking, a HFC relates to a laminar flow ofliquid, which is spatially confined within an immersion liquid (alsocalled environmental liquid). I.e., the processing liquid need beinjected via the first aperture 112 while re-aspirating liquid at thesecond aperture 114, at flow rates set so as to maintain a HFC of theinjected liquid, between apertures 112 and 114. For this to be possible,certain conditions must be fulfilled, in terms of flow rates, dimensionsof the apertures and relative distances therebetween, as known in theart.

In particular, by keeping the aspiration flow rate higher than theinjection rate, e.g., at a defined ratio, a laminar flow path ofprocessing liquid can be formed and confined within the immersion liquid60. To that aim, a minimal distance between the injection and aspirationapertures is typically between 10 μm and 10 mm, and preferably between30 μm and 2.0 mm. Also, the average diameter of the apertures 112, 114need typically be between 5 and 250 μm. The probe may otherwise compriseor connect to suitable pumping means 41-43, to generate the requiredflow rates, as known per se.

More generally though, the present bypass and control concepts may beimplemented in various types of MFP-like devices, irrespective of thedevice shape, materials used, aperture design and channel dimensions.Still, the channel and aperture diameters will typically be in themicrometers range (e.g., 5 μm to 250 μm).

For instance, suitable design parameters as used to obtain a device asshown in FIG. 11A may be taken as follows:

-   -   Bypass channel 15: 20 μm×100 μm (width×etched depth);    -   Length of bypass channel portion 151: 3 mm;    -   Length of bypass channel portion 152: 0.3 mm;    -   Injection flow rate: 1 μl/min;    -   Control flow rate: 1 μl/min;    -   Aspiration flow rate: 6 μl/min;    -   Average diameters of the channels 12, 14 and apertures 112, 114:        30 μm; and    -   Minimal gap between the injection and aspiration apertures 112,        114: 30 μm.

As illustrated in the above example, the first portion 151 has a lengththat is preferably larger than the length of the second portion 152 ofthe bypass channel 15. This too helps in achieving a larger hydraulicresistance for the first portion 151. The length ratio is preferablycomprised between 2:1 and 20:1. It may for example be of 10:1, as in theexample above.

Besides the implementation of the bypass channel 15, the design of theother channels 12, 14, 16 can be kept standard. In particular, thechannels 12, 14, 16 will preferably have, each, a hydraulic resistancethat is constant along their main channel extensions. That said, thesechannels 12, 14, 16 may have distinct resistances, as noted earlier. Forexample, the hydraulic resistance R3 of the control channel 16 may besmaller than each of the resistance R1 of the injection channel 12 andthe resistance R2 of the aspiration channel 14.

For example, the hydraulic resistances R1, R3 of the injection channel12 and the control channel 16 may be tuned to allow control of flowrates on the order of 1 μl/min, whereas the hydraulic resistance R2 ofthe aspiration channel 14 may be set to achieve flow rates on the orderof 10 μl/min. The resulting ratio (10:1) can typically be used to obtaina HFC of injected liquid. More generally, the probe and the probe headmay be configured to allow a HFC.

Several classes of embodiments can be contemplated, owing to that: (i)the bypass and control channels can be provided directly on the probehead (“on-head”) or in a distinct module (“off-head”); and (ii) the headcan be of the “vertical” or the “horizontal” type. Of particularadvantage is that each of the “on-head” and “off-head” concepts arecompatible with either type of probe head.

For instance, and as illustrated in FIGS. 1-8, and 11A-13, the bypassand control channels can be provided directly on the probe head. Namely,each of the injection channel 12, the aspiration channel 14, the bypasschannel 15 and the control channel 16 extends within a body 18 of theprobe head in that case. The bypass channel 15 fluidly connects theinjection channel 12 to the aspiration channel 14, within the body 18 ofthe head.

As it may be realized, implementing the bypass-channels directly on theMFP head allows the system to react faster in case of failure. As aresult, less processing liquid will escape the probe head andcontaminate the immersion liquid and the substrate. The “on-head”approach is compatible with both a vertical probe head (where allrelevant channels can be grooved on the same chip, as in FIGS. 11A-11C)and a horizontal probe head, as described below in detail.

In embodiments as illustrated in FIGS. 12 and 13, the MFP deviceincludes a probe head 10 f, 10 g configured as a “horizontal” probehead. The MFP device 1 f shown in FIG. 12 has a holder 2, designed forreceiving a head 10 f, the latter having a mesa 20, slightly protrudingoutwardly, so as to define a processing surface 11. Supporting posts 29are provided on the head 10 for leveling purposes. A frame 3, on top ofthe holder 2, allows the head be mounted to positioning means thatinclude, e.g., a goniometer on top (not shown), whereby the head 10 canbe positioned (vertically, along z-axis) and rotated to a preciseposition.

The device 1 f may further comprise usual equipment, such as, e.g.,tubing ports, valves, pumping means) and otherwise be configured toallow a HFC of the processing liquid.

The device 1 f may notably use a head 10 g as shown in FIG. 13. Thisprobe head 10 g can be obtained through a simple fabrication process,using a few layers 181-183. The bypass channel 15 is implemented on theupper side of a layer 182 of the head, where the routing of themicrochannels is done.

Namely, the probe head 10 g shown in FIG. 13 comprises two layers 181,182, including a control layer 182 and a routing layer 181. The bottomface of the control layer 182 covers the top face of the routing layer181. The processing surface 11 is defined by the bottom face of therouting layer 181, opposite to the top face thereof. The apertures 112and 114 are, each, defined on the bottom face of the routing layer 181.

The routing layer 181 comprises a first pair of through-vias 121, 141extending through a thickness of layer 181, so as to form respectivesegments of the liquid injection channel 12 and the liquid aspirationchannel 14. Such segments are in fluid communication with respectiveapertures 112, 114. The bypass channel 15 is defined on the top face ofthe routing layer 181.

The control layer 182 comprises a through-via (again extending through athickness of layer 182), so as to form a segment of the control channel16. The control layer 182 further comprises a second pair ofthrough-vias 122, 142 (extending through a thickness thereof), so as toform additional segments of the injection channel 12 and the liquidaspiration channel 14, respectively. After assembly of the layers 181,182, these additional segments 122, 142 make fluid communication withthe first pair of through-vias 121, 141, respectively. Using such afabrication concept, a bypass channel can easily be achieved, whichconnects channel 12, 14 (formed by respective segments 121, 122 and 141,142) as well as the control channel 16, at the interface between therouting layer 181 and the control 182 layer.

At the final stages of fabrication, additional layers may be present,such as a capping layer 183, which closes the channels on top of thecontrol layer 182. Furthermore, additional channel segments (not visiblein FIG. 13) will typically be present in the layer 182, and, e.g.,extend perpendicularly to the cutting plane of FIG. 13, so as tobring/evacuate liquid from the channel segments 122, 16, 142.

In other variants, the horizontal MFP heads can also be fabricated bymachining a block material or thanks to 3D printing (not shown).

Another class of embodiments is now described, which relies on“off-head” implementation of the bypass and control channels, inreference to FIGS. 9 and 10. The embodiments of FIGS. 9-10 assume“vertical” heads, comparable to that of FIGS. 11A-11C, except that theheads 10 c, 10 d do not comprises any bypass or control channel, whichare instead implemented in a separate module 30 c, 30 d.

Namely, the probe heads 10 c, 10 d comprise, each, a first segment 121of the injection channel 12 and a first segment 141 of the liquidaspiration channel 14. The segments 121, 141 are in fluid communicationwith the first aperture 112 and the second aperture 114, respectively.

In addition, each of the probes 1 c, 1 d comprises a bypass module 30 c,30 d, which is distinct from the probe heads 10 c, 10 d. The bypasschannel 15 and the control channel 16 are provided in the bypass module30 c, 30 d, which further comprises a second segment 122 of theinjection channel 12 and a second segment 142 of the aspiration channel14. In the bypass module 30 c, 30 d, the bypass channel 15 fluidlyconnects the second segment 122 of the injection channel 12 to thesecond segment 142 of the liquid aspiration channel 14.

Yet, the module 30 c, 30 d and the head 10 c, 10 d are arranged suchthat the channel segments 122, 142 are in fluid communication with thecomplementary segments 121, 141, respectively. This way, the bypassconcept can be implemented outside the MFP head thanks to a module 30 c,30 d that is nevertheless suitably connected to the MFP head. Thefunctionality of the bypass-channels otherwise remains the same as whenimplemented on-chip. An off-chip configuration makes the bypassfabrication independent from the MFP head, which eases the fabricationand implementation as one can rely on existing probe heads, withoutsubstantially modifying the latter. Minor modifications (e.g., to obtainthrough-vias) may nevertheless be required, depending on the availableheads.

The first and second segments of the injection and aspiration channelsmay be connected directly, assuming the head 10 d is affixed to thebypass module 30 d, as in FIG. 10, thanks to through-vias, which ensurefluid communication. That is, in FIG. 10, the probe head 10 d comprisesthrough-vias (denoted by black disks), extending transversely to themain plane of the chip (i.e., corresponding to layer 21 in FIGS.11A-11C), from the end of the segments 121, 141. Correspondingthrough-vias (not visible) extend, in vis-à-vis, from a surface of themodule 30 d onto which the chip is fixed, toward channel segments 122,142 provided in the bypass module 30 d, so as to ensure fluidcommunication with corresponding channel segments 121, 141 on the chip,respectively.

A capping layer (not shown) comes to close the channel segments 121,141. Similarly, a capping layer may close channel segments grooved on abody of the module 30 d. This additional capping layer may be providedon either side of this body and may need to comprise though-vias ifintercalated between the head 10 d and this body. In variants, themodule 30 d is obtained by 3D printing, with channels segments extendingwithin the body of the module. Through-vias would, again, be involved toensure proper fluid communication.

In variants to FIG. 10, intermediate channel segments 12 t, 14 t may beused, e.g., provided as flexible tubes, as in FIG. 9, to connect channelsegments 122, 142 in the bypass module 30 to the corresponding, on-chipchannel segments 121, 141. Such variants allow existing concepts ofvertical heads to be re-used, without any modification thereto. Thevariant of FIG. 10 allows somewhat faster reactivity, compared to FIG.9, owing to the increased proximity of the bypass channel with thechannel segments 121, 141. Yet, a substantially faster reactivity can beobtained if the bypass and control channels are both implementeddirectly at the probe head, as noted earlier.

Vertical probe heads 10, 10 a-10 e as shown in FIGS. 1-11C areparticularly simple to fabricate. They may for instance be fabricatedfrom two layers 21, 22 of material (e.g., silicon and glass,respectively), as illustrated in FIG. 11C. When used together with abypass module, then only the channel segments 121, 141 need be groovedon a layer 21 (e.g., a silicon chip) of the head and closed by anotherlayer 22 (e.g., glass). Thus, already existing concepts of verticalheads can advantageously be re-used, without the additional bypass andcontrol channels need thereon. In the “on-head” approach, all channels12, 14, 15, 16 can be grooved on layer 21, as assumed in FIGS. 11A-11C.

For completeness, we note that, although vertical probe heads areassumed in FIGS. 9-10, the off-head concept can equally be used withhorizontal heads. Multilayer devices are typically relied upon in thatcase, as in FIG. 13, which involve materials such as, silicon, glass,PDMS or other elastomers, hard plastics, etc., as usual in the art.

At present, referring to FIGS. 7, and 14A-14B, further embodiments aredescribed, which concern a microfluidic probe 1 a, wherein theprocessing surface 11 of the head 10 a comprises multiple aspirationapertures 114, 114 a, 114 b. As better seen in FIGS. 14A-14C, a set oftwo (FIG. 7, 14A or more (FIG. 14B) apertures 114, 114 a, 114 b may beprovided on the processing surface 11, where each aspiration aperture isarranged at a distance from the injection aperture 112. Consistently,and as depicted in FIG. 7, the probe 1 a comprises a corresponding setof aspiration channels 14, 14 a, which extend from a respective one ofthe apertures 114, 114 a.

In such a case, additional bypass channels may advantageously beprovided, as illustrated in FIG. 7, so as to avoid leakage of theprocessing liquid from any of the apertures. I.e., several bypasschannels 15, 15 a are provided in the probe, so as to fluidly connectthe liquid injection channel 12 to a respective one of the aspirationchannels 14, 14 a. Consistently, two or more control channels 16, 16 afluidly connect to respective bypass channels 15, 15 a.

Providing additional aspiration apertures as well as correspondingaspiration, bypass and control channels can be exploited to avoidleakage of the processing liquid into the immersion liquid. E.g., incase a topographical variation on the processed surface S startsblocking a given one 14 a of the aspiration apertures (as illustrated inFIG. 7), another aspiration aperture 14, e.g., symmetrically positionedwith respect to the blocked aperture 14 a, may still ensure liquidaspiration, while excess of processing liquid injected from channel 12can be diverted through the bypass 15 a, as indicated by the curvedarrow in FIG. 7. If all apertures happen to be blocked (not shown), thenthe processing liquid will be suitably diverted through both bypasschannels 15, 15 a, to avoid leakage of processing liquid.

FIG. 14A assumes symmetric (square) aspiration apertures. In variants tosymmetric openings, one may use a curved slit or a set of curved slitsfor aspiration, as discussed below, in reference to FIGS. 14B, 14C, 15and 16.

In embodiments such as depicted in FIGS. 14B-16, the probe headcomprises one or more aspiration slits 114, 114 a, 114 b, each shaped soas to partly extend around the first aperture 112 on the processingsurface 11. As a result, the injection aperture 112 is not completelysurrounded by the slit(s) on the processing surface 11, which istypically defined on a mesa 20 of the probe head 10 h, see FIG. 15. Eachslit is partly coiled around the injection aperture (it may be eitherbent or curved).

Because the aspiration slit(s) extend(s) partly around the injectionaperture, a degree of confinement of the injected liquid can beobtained, in normal operation of the head (assuming no failure). Thatis, injected liquid remains confined due to liquid aspirated at theslit, which forms a barrier extending around the injected liquid. Theliquid barrier created by the liquid aspiration helps to improvehomogeneity in the deposited liquid or particles thereof, such as cells.Meanwhile, the shape of the slit allows immersion liquid in the vicinityof the head to be aspirated via the slit. This further allows the flowvelocity of the injected liquid to be set partly (if not essentially)independent from the aspiration flow, which, in turn, eases theoperation of the head.

Note that, in that case, the bypass channel 15 may be partly circular,or, more generally, shaped, so as to ease fluid communication from theinjection channel to the aspiration channel. Such a bypass channel maybe provided at an interface between two layers, as in FIG. 13.

In other variants, such as depicted in FIG. 8, the probe 1 b comprisesseveral bypass channels 15, 15 a, 15 b, which all connect the same twochannels 12, 14. That is, each of the bypass channels 15, 15 a, 15 b isarranged, e.g., directly in the probe head 10 b, so as to fluidlyconnect the injection channel 12 to the aspiration channel 14. Inembodiments such as depicted in FIG. 8, the probe further comprises aplurality of corresponding control channels 16, 16 a, 16 b, whichfluidly connect to a respective one of the bypass channels 15, so as toform respective junctions therewith. To that aim, the probe 10 b may beagain configured as a multilayer probe head. Only the apparent,front-most layer is depicted in FIG. 8, for clarity, onto which isgrooved the control channel 16. Additional control channels 16 a, 16 b(denoted by dashed lines in FIG. 8) can be defined at (hidden)interfaces between any two contiguous layers and connect to respectivebypass channels 15 a, 15 b, thanks to through-vias (denoted by dashedcircles in FIG. 8). Although a vertical head is assumed in FIG. 8, wenote that the same concept can be implemented in a horizontal head.

Having multiple bypass channels allows gradual diversion of theprocessing liquid, when necessary. It further allows the device to havedifferent working points, i.e., different bypass thresholds can be set,which makes it possible to cope with different failure scenarios with asame device, while operating the latter in a fully passive mode.

In variants (not shown), only one control channel is needed, whichcrosses all bypass channels, so as to further connect each of theadditional bypass channels 15 a, 15 b. That is, a same control channelfluidly connects to each bypass channel in that case. Such variantstypically require to adapt hydraulic resistances of the additionalportions of the control channel.

Referring back to FIG. 1, the present probes may, in embodiments, beconfigured to operate in a constant liquid flow mode, and/or in constantpressure mode. That is, the system may be configured to operate in onlyone of these two modes, or in each of these two modes, it being notedthat the probe is normally operated in one mode at a time. In constantliquid flow mode, a constant liquid flow is applied to each of theinjection channel 12, the aspiration channel 14 and the control channel16. In constant pressure actuation mode, a constant pressure is appliedto each of these channels 12, 14 16. Applying a constant flowrate/pressure to the control channel 16 allows the probe to be passivelyoperated, such that no active control and dynamic adaptation isrequired, even in case of failure. The system is fully passive asconstant flow rates or pressures are applied to each of the channels 12,14, 16.

To that aim, the present probe systems may include pressure sources 41,42, and a vacuum source 43, as depicted in FIG. 1. In addition, liquidtanks (not shown) may be present, as usual in the art.

Moreover, a check valve (or a proportional valve) 44, and a flow sensor45 may optionally be involved, to enable active or semi-active control,as assumed in FIG. 1. More generally, other active control means may beinvolved. In other variants, one may combine active and passive controlsto enable additional functionality. For example, passive flow/pressurecontrol in the bypass channel may be used to provide a sensitivefeedback signal, which can be measured by a flow sensor 45 in the bypasssupply line. This notably allows detection of an excessively large gapdistance, tilt variations and blockages of apertures. Also, an activecontrol element, i.e. a switch valve, in the bypass supply line may beused to make it possible to adjust, or even fully suppress the HFCwithout changing the injection and aspiration flow rates in theinjection and aspiration channels 12 and 14. This proves to be veryadvantageous in applications where both a fast switching of liquids anda continuous flow of sample in the injection channel 12 are needed.

Referring to FIGS. 1, 3-6, and 15-16, another aspect of the invention isnow briefly described, which concerns methods of operating a probe 1, 1a-1 f such as described herein. Aspects of such methods have implicitlybeen described in reference to the devices 1, 1 a-1 f or theircorresponding heads 10, 10 a-10 h. Essentially, such methods revolvearound positioning the head 10, 10 a-10 h in proximity with a samplesurface S to be processed (so as for the processing surface 11 to facethe sample surface 5), and injecting processing liquid via an injectionaperture while aspirating liquid from one or more aspiration apertures,to process the sample surface.

As described earlier, the sample surface S is typically immersed in animmersion liquid 60, so as for the probe head 10, 10 a-10 h to be atleast partly immersed in the immersion liquid 60. In addition, theprobes are preferably operated so as to maintain a hydrodynamic flowconfinement of injected liquid between the injection aperture and theaspiration aperture(s).

The MFP head can either be kept static with respect to the samplesurface 5, while depositing the processing liquid (e.g., containingcells), to obtain a homogeneous deposition, deposited as a spot onto thesample surface S. In variants, the MFP head can be scanned across thesample surface 5, e.g., to obtain a pattern, as discussed below andillustrated in FIG. 16.

For example, one may use an aspiration aperture shaped as a curvedaspiration slit, as in FIG. 14C. In that case, and as illustrated inFIG. 16, the partial extension of the aspiration aperture slit aroundthe injection aperture gives rise to a gap in the aspiration slit. Thus,the head can be scanned in a direction opposite to the gap with the gaplocated on the trailing edge, so as to minimize perturbations to thepattern of deposited cells. As further seen in FIG. 16, the head 10 h isfirst scanned from left to right and then from bottom to top. Forexample, red blood cells 50 can be deposited onto the substrate duringthe scanning. The deposition of the cells can be performed over largedistances (e.g., of 190 μm) and with a scanning velocity of, e.g., 50 μmper second. The device shown in FIG. 16 comprises a ring-shapedprotruding structure of 30 μm high.

As previously described in reference to FIGS. 3-6, the present probes 1,1 a-1 f may advantageously be used to cope with various failurescenarios. In particular, if one or each of the injection and aspirationapertures 112, 114 (114 a, . . . ) happen to be blocked, due to theproximity of the probe head with the sample surface S, then liquidinjected via the channel 12 can be (passively) diverted through thebypass channel 15 to be aspirated via the aspiration channel(s).

In other cases, e.g., when the distance to the surface of the samplebecomes too large (FIG. 6), the liquid flowing in the control channel 16can be (passively) diverted to flow through the injection aperture 112.Hence, the flow through the injection aperture 112 increases and thevolume occupied by the injected liquid in the space between the probe 1and the surface S protrudes further downwards and partially compensatesfor too large operating distances 6 a.

In variants to passive operations, the liquid flow rate or the liquidpressure may be adjusted in the control channel 16, to provide(semi-)active control. This assumes that the liquid flow (or thepressure) is monitored in one or each channel 12, 14. Thus, if a liquidflow (or pressure) variation is detected, which is indicative of afailure, then flow/pressure can be adjusted in the control channel 16,as needed to compensate for the failure detected.

To that aim, a shut-off valve may be involved in the flow path of thecontrol channel 16. This notably allows fast on/off switching of a HFC,without noticeably interrupting the injection liquid flow, and, in turn,improves the switching speed and stability of the HFC.

In addition, the footprint of the HFC can be varied by changing the flowrate/pressure in the control channel 16, as the overall injection toaspiration ratio changes (this ratio is defined as the sum of theinjection flow and the control flow rate, divided by the aspiration flowrate). Such added flexibility may be exploited to generate patterns,modulate shear stress, or to reduce requirements on infrastructure. Thatis, instead of two high precision flow control lines for injection andaspiration, the latter two can be set coarsely and only one fine controlis needed to set the exact shape of the HFC via the flow through thecontrol channel 16.

In the simpler, passive solutions described earlier, the transitionbetween normal operation and failure mode is typically set ex-ante,i.e., the transition threshold is set to a desired level byappropriately adjusting the flow/pressure in the control channel 16,i.e., prior to operate the probe. Then, the liquid flow rate (or theliquid pressure) is kept constant in the control channel 16 whileprocessing the sample.

While the present invention has been described with reference to alimited number of embodiments, variants and the accompanying drawings,it will be understood by those skilled in the art that various changesmay be made and equivalents may be substituted without departing fromthe scope of the present invention. In particular, a feature(device-like or method-like) recited in a given embodiment, variant orshown in a drawing may be combined with or replace another feature inanother embodiment, variant or drawing, without departing from the scopeof the present invention. Various combinations of the features describedin respect of any of the above embodiments or variants may accordinglybe contemplated, that remain within the scope of the appended claims. Inaddition, many minor modifications may be made to adapt a particularsituation or material to the teachings of the present invention withoutdeparting from its scope. Therefore, it is intended that the presentinvention not be limited to the particular embodiments disclosed, butthat the present invention will include all embodiments falling withinthe scope of the appended claims. In addition, many other variants thanexplicitly touched above can be contemplated. For example, othermaterials than silicon or glass can be contemplated for layers 21, 22,such as, e.g., PDMS or other elastomers, hard plastics (e.g., PMMA, COC,PEEK, PTFE, etc.), ceramics, or stainless steel.

What is claimed is:
 1. A method of operating a probe, the probecomprising: a probe head with a processing surface that comprises afirst aperture and a second aperture; a liquid injection channel, whichleads to the first aperture; a liquid aspiration channel, which extendsfrom the second aperture, the liquid aspiration channel configured toaspirate liquid via the second aperture; a bypass channel, arranged soas to fluidly connect the liquid injection channel to the liquidaspiration channel; and a control channel, the control channel beingconfigurable to match a pressure at a junction of the liquid injectionchannel and the bypass channel and a pressure at a junction of thecontrol channel and the bypass channel and which fluidly connects to thebypass channel, hence forming a junction therewith, so as to define twoportions of the bypass channel, the portions including: a first portionthat extends from said junction to the liquid injection channel; and asecond portion that extends from that same junction to the liquidaspiration channel, wherein the method comprises: positioning the probehead in proximity with a sample surface to be processed, so as for theprocessing surface to face the sample surface; adjusting a pressure inthe control channel during a first operation to match the pressure atthe junction of the liquid injection channel and the bypass channel andthe pressure at the junction of the control channel and the bypasschannel and adjusting the pressure in the control channel during anotheroperation to enable liquid to pass from the liquid injection channelthrough the bypass channel to the liquid aspiration channel; andinjecting processing liquid via the first aperture while aspiratingliquid from the second aperture, to process the sample surface.
 2. Themethod according to claim 1, wherein the method further comprises:blocking one or each of the first aperture and the second aperture, dueto a proximity of the probe head with the sample surface processed,whereby processing liquid injected via the liquid injection channelpasses through the bypass channel to get aspirated via the liquidaspiration channel.
 3. The method according to claim 1, wherein themethod further comprises one of: adjusting a liquid flow rate in thecontrol channel; and adjusting a liquid pressure in the control channel.4. The method according to claim 3, wherein adjusting the liquid flowrate or the liquid pressure in the control channel is performed prior topositioning the probe head in proximity with the sample surface, theliquid flow rate or the liquid pressure in the control channel beingthen kept constant while injecting processing liquid via the firstaperture and aspirating liquid from the second aperture to process thesample surface.
 5. The method according to claim 1, wherein: the probehead is positioned in proximity with the sample surface, onto which isan immersion liquid, so as for the probe head to be at least partlyimmersed in the immersion liquid; and liquid aspirated from the secondaperture comprises immersion liquid.
 6. The method according to claim 5,wherein the steps of injecting the processing liquid and aspiratingliquid are performed so as to maintain a hydrodynamic flow confinementof injected liquid between the first aperture and the second aperture.